Thiopeptide compounds

ABSTRACT

A microorganism belonging to the genus  Bacillus  is cultured and thiopeptide compounds QN3323 and thiocillin I/II compounds are collected from the culture medium. The compounds are useful as drugs to treat infections caused by multidrug-resistant bacteria.

TECHNICAL FIELD

The present invention relates to remedies for infections withmultidrug-resistant bacteria such as MRSA and VRE, which comprise athiopeptide compound as the active ingredient thereof. It also relatesto novel thiopeptide compounds QN3323 and their production, and to drugsthat contain any of the compounds as an active ingredient thereof.

Heretofore well known are various antibiotics that are produced bymicroorganisms, including, for example, β-lactam antibiotics such aspenicillin, cephalosporin, carbapenem; macrolide antibiotics such aserythromycin, josamycin, rokitamycin; and aminoglycoside antibioticssuch as kanamycin, gentamycin, tobramycin, etc. Some of theseantibiotics and chemically modified derivatives thereof are extremelyeffective as bactericides and antibacterials and many of them are nowactually in clinical use.

Recently, however, methicillin-resistant Stayphylococcus aureus(hereinafter referred to as MRSA) having high resistance to theseantibiotics has frequently appeared in the field of clinical medicine,and it is now a serious social problem. In 1996, in addition, it wasreported in Japan that enterococci highly resistant to vancomycin, thathas been widely used to treat infections caused by MRSA(vancomycin-resistant Enterococcus—hereinafter referred to as VRE), wereisolated from a clinical case. Apart from these, some types ofPseudomonas aeruginosa and Klebsiella pneumoniae are known to have highresistance to various drugs. These bacteria are referred to asmultidrug-resistant bacteria, and are now specifically problematic inthe field of clinical medicine. Therefore, it is much desired to developantibiotics that are effective against such multidrug-resistantbacteria.

Macrocyclic compounds having a thiazole as the ring constitutivecomponent are generically referred to as thiopeptide compounds, andvarious compounds of this type have been reported. They differ, forexample, in the number of the ring-constituting atoms in them and in theelements that constitutes their rings. Concretely, there are reportedmonocyclic macrocyclic-structured compounds such as thiocillin I, andtricyclic macrocyclic-structured compounds such as glycothiohexide-α (J.Antibiotics 47: 894, 1994), and they differ in their ring structures.Thiocillin I, thiocillin II and micrococcin P2 are compounds having amonocyclic macrocyclic structure in common, and their antibacterialactivity against gram-positive bacteria has been reported (J.Antibiotics 29: 366–374, 1976; J. Chem. Soc., Chem. Commun., 1978(6),256–8; International Patent Publication WO97/48408). However, no reporthas heretofore been made until now relating to the antibacterialactivity of these compounds against multidrug-resistant bacteria such asMRSAT and VRE.

Regarding thiopeptide compounds, anti-MRSA or anti-VRE activity ofglycothiohexide α (J. Antibiotics 47:894, 1994), amythiamicin A (J.Antibiotics 47: 668, 1994; ibid. 47:1153, 1994), sulfomycin (J. Nat.Prod., 62:1562, 1999) and promoinducin (Biosci. Biotechnol. Biochein.59:876, 1995) has been reported. Glycothiohexide α is a compound havinga tricyclic macrocyclic structure. Amythiamicin A, sulfomycin andpromoinducin have a monocyclic macrocyclic structure, but differ fromthe above-mentioned thiocillins in the type of amino acids thatconstitute their rings, and, in addition, the number of atoms thatconstitute the ring of the former macrocyclic compounds is larger thanthat of thiocillins.

We, the present inventors have conducted research to identifyantibiotics produced by naturally existing microorganisms, and, as aresult, have found that cells of a type that belong to the genusBacillus may produce compounds having an antibacterial activity againstbacteria that include multidrug-resistant bacteria such as MRSA and VRE.Further, we have studied in detail the culture of the cells and havesucceeded in isolating novel thiopeptide compounds QN3323-A, QN3323-Band QN3323-Y1 and known thiopeptide compounds thiocillin I, thiocillinII and micrococcin P2 from the culture of the cells, and, in addition,have found that these compounds have anti-MRSA activity and anti-VREactivity, and have completed the present invention.

Specifically, the invention relates to remedies for infections withmultidrug-resistant bacteria, especially those for infections with MRSAor VRE, which contain, as an active ingredient thereof, a thiopeptidecompound of the following formula (I) or a pharmaceutically acceptablesalt thereof.

wherein A represents CH—OH or C═O; R represents H or CH₃; R′ representsH or OH.

The invention also relates to a method for producing the thiopeptidecompounds, which comprises cultivating cells of Bacillus sp. QN03323(FERM BP-7864) that belongs to the genus Bacillus followed by collectingthe thiopeptide compounds of formula (I) from the culture of the cells.

The invention further relates to novel thiopeptide compounds of thefollowing formula (I′) or their pharmaceutically acceptable salts:

wherein A represents CH—OH or C═O; R represents H or CH₃; R^(a) andR^(b) each represent H or CH₃;provided that when R^(a) is H, then R^(b) is CH₃; when R^(a) is CH₃,then R^(b) is H; when A is CH—OH, then R^(a) is H and R^(b) is CH₃.

Preferred are the following compounds:

(1) A is C═O; R is H; R^(a) is CH₃; and R^(b) is H (QN3323-A),

(2) A is C═O; R is CH₃; R^(a) is CH₃; and R^(b) is H (QN3323-B), and

(3) A is CH—OH; R is H; R^(a) is H; and R^(b) is CH₃ (QN3323-Y1).

The invention still further relates to a method for producing thethiopeptide compounds, which comprises cultivating microorganismsbelonging to the genus Bacillus and having the ability to produce thethiopeptide compounds of formula (I′), followed by collecting thethiopeptide compounds from the culture of the cells.

The invention still further relates to a medicine, especially anantibacterial agent that contains, as the active ingredient thereof, thethiopeptide compound of formula (I′) or its pharmaceutically acceptablesalt.

The invention is described in detail hereinunder.

The thiopeptide compounds (I) of the invention are obtained by anordinary method that comprises cultivating cells belonging to the genusBacillus and having the ability to produce the compounds in a nutrientmedium followed by collecting the compounds from the culture where thecompounds have accumulated. The microorganisms that are used in themethod of producing the compounds may be any that belong to the genusBacillus and have the ability to produce the compounds. One example ofthe microorganisms of the type useful for the method is a gram-positivestrain QN03323 belonging to the genus Bacillus, which was separated fromunidentified sponge collected at the seaside of Taketomi-cho,Yaeyama-gun, Okinawa-ken (Iriomote Island). The bacterial properties ofthe strain are as follows:

1) Morphological Property:

This strain is a gram-positive bacillus and is motile. The size of thecell is from 1 to 1.7×5 to 8 μm. One cell has one oval spore formedtherein.

2) Cultural Property:

This strain forms white colonies on a meat extract agar medium. Eachcolony is circular, its surface is not glossy, and its periphery isrough. In static culture in meat extract broth, the culture with thecells growing therein is completely cloudy or the cells sink down, andno film is formed on the surface of the medium. In stab culture in brothgelatin, the cells liquefy the gelatin. In culture with litmus milk, theculture is peptonized and is neutral, and this is not coagulated.

3) Physiological Property:

The physiological property of QN03323 is as follows:

Nitrate positive Dinitrification negative Reduction MR Test negative VPTest positive Indole negative Hydrogen negative Production SulfideProduction Starch positive Citric Acid negative Hydrolysis UtilizationNitrate negative Ammonium Salt negative Utilization UtilizationWater-Soluble negative Urease positive Fluorescent Dye ProductionOxidase positive Catalase negative Growing 10 to 45° C. Optimum Growing28 to 40° C. Temperature Temperature Range pH Range for pH 5 to 9Optimum pH for pH 6 to 8 Growth Growth Growth under positive Argininepositive Anaerobic Dihydrolase Condition Growth in 3% NaCl-addedpositive broth medium Growth in 6% NaCl-added positive broth medium(Production of Acid from Saccharide)

L-arabinose negative D-galactose negative D-xylose negative Maltosepositive D-glucose positive Trehalose positive D-mannose positiveLactose negative D-fructose positive D-sorbitol negative Sucrosepositive Glycerin positive Inositol negative Starch positive D-mannitolnegative

The above-mentioned mircrobial properties of the strain are summarized.The strain is a gram-positive facultative anaerobic bacillus, and ismotile. Its growth temperature range falls between 10 and 45° C.; it ispositive in nitrate reduction, starch hydrolysis, urease, oxidase test,gelatin liquefying reaction and arginine dihydrolase; and it producesacid from D-glucose, D-mannose, D-fructose, sucrose, maltose, trehalose,glycerin and starch. On the other hand, it is negative indenitrification, indole production, hydrogen sulfide production, citricacid utilization, inorganic nitrogen source utilization and catalasetest.

Based on the above-mentioned properties thereof, the strain was lookedup in BERGEY's Manual of Systematic Bacteriology, 1989 and otherreferences, and, as a result, it was identified as a type of bacteriabelonging to the genus Bacillus, and was named Bacillus sp. QN03323.

This strain, Bacillus sp. QN03323, has been deposited with the NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary Center (Tsukuba Central 6, 1—1—1, Higashi,Tsukuba, Ibaraki, Japan, 305-8566) as FERM BP-7864 (as of depositiondate, Feb. 14, 2001). In general, microorganisms may artificially ornaturally mutate. Therefore, Bacillus sp. QN03323 for use in theinvention shall include not only the microorganisms separated fromnatural matters but also those artificially mutated from them throughexposure to UV rays, radiations or chemicals and their natural mutants.

(Production Method)

The compounds of the invention may be obtained by cultivating themicroorganisms belonging to the genus Bacillus and having the ability toproduce the compounds of the invention. The cultivation of themicroorganisms may be effected in any ordinary manner of cultivatingordinary microorganisms.

The medium to be used for the cultivation may be any one that containsnutrient sources for the microorganisms having the ability to producethe compounds of the invention (e.g., Bacillus sp. QN03323). Forexample, it may be any of synthetic media, semi-synthetic media ornatural media. Regarding the composition of the medium, the carbonsource thereof includes, for example, L-arabinose, D-xylose, D-glucose,D-fructose, sucrose, inositol, L-rhamnose, raffinose, D-mannitol,mannose, melibiose, lactose, D-galactose, maltose, trehalose, salicin,xanthine, chitin, starch, glucose, dextrin, glycerin, and vegetable oil;and the nitrogen source thereof includes, for example, broth, peptone,gluten meal, cotton seed refuse, soybean meal, peanut meal, fish meal,corn steep liquor, dry yeast, yeast extract, ammonium chloride, ammoniumsulfate, ammonium nitrate, uric acid and other organic and inorganicnitrogen compounds. Metal salts may be optionally present in the medium,including, for example, sodium, potassium, magnesium, calcium, zinc,iron or cobalt sulfates, nitrates, carbonates and phosphates.

Further if desired, the medium may also contain growth promoters ordefoaming agents such as methionine, cysteine, cystine, thiosulfates,methyl oleate, lard oil, silicone oil and surfactant.

Preferably, the cells are cultivated under an aerobic conditions. Thetemperature for cultivation may fall between 10 and 45° C., preferablybetween 25 and 30° C. or so. The pH of the medium may fall approximatelybetween 5 and 9, preferably approximately between 6 and 8 for betterresults. The cultivation period can be suitably determined depending onthe composition of the medium used and the temperature for cultivation.In general, it may be approximately from 1 to 20 days, preferablyapproximately from 2 to 5 days.

For isolating and purifying the compounds of the invention from theculture, any ordinary method of isolating and purifying physiologicalsubstances from ordinary microbial cultures can be employed. Forexample, the intended compounds are extracted out of the culture with asuitable solvent, and the effective substances are isolated and purifiedfrom the resulting extracts. Concretely, based on the antibacterialactivity thereof as the index for isolation and purification thereof,the compounds of the invention may be isolated and purified by anyordinary method that is used in producing ordinary physiologicallyactive substances by utilizing the solubility and the solubilitydifference of the substances in suitable solvents. These methods may beused singly, if desired, or may be combined in any desired manner forrepetition.

The compounds of the invention may form acid addition salts, and thesalts are pharmaceutically acceptable salts. Concretely, they includeacid-addition salts with inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid,phosphoric acid; or with organic acids such as formic acid, acetic acid,propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid,maleic acid, lactic acid, malic acid, tartaric acid, citric acid,methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid.The salts of the compounds of the invention may be obtained in anyordinary salt formation from the compounds of the invention that havebeen isolated and purified or during the stage of isolation andpurification of the compounds from the cultures.

The thiopeptide compounds (I) or their pharmaceutically acceptable saltsof the invention have an asymmetric carbon and a double bond, andtherefore include optical isomers and geometrical isomers based onthese. The compounds of the invention encompass mixtures of theseisomers and isolated isomers. Further, the invention also encompassesvarious hydrates and solvates of the thiopeptide compounds (I) or theirpharmaceutically acceptable salts, as well as their crystals and crystalpolymorphic substances. The compounds of the invention includepharmaceutically acceptable prodrugs thereof. The pharmaceuticallyacceptable prodrugs are compounds that have a group capable of beingconvened into OH or the like in the compounds of the invention throughsolvolysis or under physiological conditions. The groups capable offorming such prodrugs are described in, for example, Prog. Med., 6,2157–2161 (1958); Pharmaceutical Research and Development (by HirokawaPublishing Company, 1990), Vol. 7, Drug Design, 163–198.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹H-NMR chart of QN3323-Y1 in DMSO-d₆; and

FIG. 2 is a ¹³C-NMR chart of QN3323-Y1 in DMSO-d₆.

INDUSTRIAL APPLICABILITY

The compounds (I) of the invention have antibacterial activity againstmultidrug-resistant bacteria such as MRSA and VRE, and are thereforeuseful as remedies for infections with multidrug-resistant bacteria,especially for infections with MRSA or VRE. The novel compounds (I′) ofthe invention are useful as active ingredients for pharmaceuticalpreparations. In particular, since they have antibacterial activity,they are useful as antibacterial agents.

The pharmaceutical preparations that contain, as the active ingredientthereof, one or more of the compounds or their salts of the inventionmay be prepared by the use of carriers, vehicles and other additivesthat are generally used in formulating ordinary pharmaceuticalpreparations.

The administration route for the preparations may be any of oraladministration with tablets, pills, capsules, granules, powders orliquids, or parenteral administration with intravenous or intramuscularinjections, suppositories, subcutaneous preparations, nasal preparationsor inhalations.

The solid composition for oral administration in the invention includestablets, powders and granules. In such solid compositions, one or moreactive substances are mixed with at least one inert vehicle such aslactose, mannitol, glucose, hydroxypropyl cellulose, microcrystallinecellulose, starch, polyvinylpyrrolidone, magnesium metasilicatealuminate. In an ordinary manner, the compositions may contain any inertadditives, for example, a lubricant such as magnesium stearate adisintegrator such as sodium carboxymethyl starch, and a dissolutionpromoter. Optionally, the tablets and the pills may be coated with asugar film or with a gastric-soluble or intestinal-soluble coatingagent.

The liquid compositions for oral administration may includepharmaceutically acceptable emulsions, liquids, suspensions, syrupds,elixirs, as well as any other ordinary inert solvent such as pure waterand ethanol. Apart from such an inert solvent, the composition mayfurther contain auxiliary agents such as solubilizers, wetting agents,suspending agents, as well as sweeteners, flavorings, aromas, andpreservatives.

The injections for parenteral administration contain germ-free aqueousor non-aqueous liquids, suspensions or emulsions. The aqueous solventmay be, for example, distilled water for injections and physiologicalsaline. The non-aqueous solvent includes, for example, propylene glycol,polyethylene glycol, vegetable oil such as olive oil, alcohols such asethanol, and Polysorbate 80 (trade name). These compositions may furthercontain isotonizers, preservatives, wetting agents, emulsifiers,dispersants, stabilizers and dissolution promoters. These aresterilized, for example, through filtration through bacteria-trappingfilters, or through addition of bactericide thereto, or throughirradiation. As the case may be, germ-free solid compositions areprepared, and they may be dissolved or suspended in germ-free water orgerm-free solvent for injections before use.

The dose of the active ingredient compound in the invention may besuitably determined, depending on the condition, the age and the sex ofthe cases to which it is administered, but is, in general, approximatelyfrom 0.1 to 1000 mg/adult/day for oral administration, and isapproximately from 0.1 to 500 mg/adult/day for parenteraladministration. This may be administered all at a time or in multipletimes. The dose shall vary depending on various conditions.

BEST MODES OF CARRYING OUT THE INVENTION

The invention is described in more detail with reference to thefollowing Examples. The compounds of the invention should not be limitedto those in the following Examples.

EXAMPLE 1

A medium containing 10 g of glucose, 20 g of potato starch, 5 g ofpolypeptone, 5 g of yeast extract, 4 g of calcium carbonate and 1 literof distilled water (having a pH of 7.0 before sterilization) wasprepared, put into 500-ml Erlenmeyer flasks with 100 ml each therein,and sterilized at 121° C. for 20 minutes. The cells of Bacillus sp.QN03323 that had been grown on a Bennett-agar medium were scraped outand inoculated onto the medium, and cultivated with shaking at 220 rpmat 28° C. for 3 days to prepare a seed culture. Next, a mediumcontaining 5 g of glucose, 5 g of Bactopeptone (by Difco), 2 g of yeastextract and 1 liter of distilled water (having a pH of 7.5 beforesterilization) was prepared and put into 500-ml Erlenmeyer flasks with100 ml each therein. 100 flasks with the medium therein were thusprepared, and these were sterilized at 121° C. for 20 minutes. 2 ml ofthe seed culture was inoculated onto the medium of every flask, andcultivated with shaking at 220 rpm at 28° C. for 3 days.

Thus cultivated, 10 liters of the culture was controlled to have a pH of7.0, to which was added 40 liters of acetone. The resulting extract waswell stirred, and filtered, and acetone was removed from it underreduced pressure. 12 liters of ethyl acetate was added to it, and wellstirred, and the ethyl acetate layer was concentrated under reducedpressure. Then, ethyl acetate was removed from it. The residue wassubjected to column chromatography with Sephadex LH-20 (by AmershamPharmacia Biotec, φ30 ×350 mm), and developed withN-hexane/dichloromethane/methanol (4/5/1) to obtain an active fraction.The active fraction was subjected to silica gel thin-layerchromatography (with Kieselgel 60F254, 20 mm×20 mm, by Merck), developedwith chloroformlmethanol (15/1), and the active fraction was scrapedout. The active fraction was then subjected to ODS column chromatography(φ20×250 mm, Develosilpack, by Nomura Chemical), and an active substanceto give a peak was fractionated with an elution solvent of aqueous 70%methanol solution, at a flow rate of 9.0 ml/min and a detectionwavelength of 210 nm. From it, the compounds of the invention, QN3323-A(5.0 mg) and QN3323-B (1.5 mg) were isolated. Similarly, knowncompounds, thiocillin I (11 mg) and thiocillin II (5.3 mg) wereisolated.

EXAMPLE 2

100 liters of a culture that had been obtained in the same manner as inExample 1 was centrifuged, and the supernatant was subjected to columnchromatography with Diaion HP-20 (by Mitsubishi Chemical) (10 liters).The deposit was extracted with aqueous 80% acetone solution (25 liters)and concentrated under reduced pressure to remove acetone. Then, thiswas again subjected to column chromatography with Diaion HP-20 as above.The HP-20 column was washed with water (30 liters) and aqueous 30%acetone solution (30 liters), and then eluted with aqueous 90% acetonesolution (30 liters). The eluate fraction was concentrated under reducedpressure to remove acetone through evaporation. The resultingconcentrate was extracted twice with 10 liters of ethyl acetate, and theethyl acetate layer was concentrated to dryness under reduced pressure.The resulting solid was then subjected to silica gel columnchromatography (with Kieselgel 60 by Merck, φ65 mm×130 mm), and elutedwith chloroform/methanol (9/1 and 5/1) solutions. The active fractionwas concentrated, and then subjected to middle-pressure silica gelcolumn chromatography (ULTRAPACK SI-40C by Yamazen, φ37 mm×300 mm), andeluted with chloroform/methanol (20/1) solution to fractionate activefractions. This contained QN3323-A (91.7 mg), QN3323-B (27.3 mg),QN3323-Y1 (161 mg), thiocillin I (870 mg) and thiocillin II (305.5 mg).

The active fraction obtained through the middle-pressure silica gelcolumn chromatography was further subjected to ODS HPLC (Develosil C30UG-5 by Nomura Chemical, φ20 mm×250 mm), and eluted with aqueous 50%acetonitrile solution to fractionate active fractions. This ismicrococcin P2 (4.1 mg).

Of the compounds that had been extracted, purified and isolatedaccording to the process mentioned above, the physical data of thecompounds of the invention QN3323-A, QN3323-B and QN3323-Y1 are given inTables 1 to 3 and FIGS. 1 and 2. In addition, the physical data of theother isolated thiocillin I, thiocillin II and micrococcin P2 weremeasured, and it was confirmed that the compounds isolated herein werethe same as the known compounds from those data.

TABLE 1 Physicochemical properties of QN3323-A and QN3323-B QN3323-AQN3323-B QN3323-Y1 Property Colorless powder Colorless powder Meltingpoint (dec.) >220° C. >220° C. [α]_(D) ²⁵ (MeOH) 64.7° (c0.37) 60.9° (c0.15) Molecular formula C₄₈H₄₇N₁₃O₁₀S₆ C₄₉H₄₉N₁₃O₁₀S₆ C₄₈H₄₉N₁₃O₁₀S₆Mass Spectroemtry: ESI-MS (M + H)⁺ 1158 1172 1160 TOFHR-MS Found (m/z)1180.1795 1194.1933 1160.2130 (M + Na)⁺ (M + Na)⁺ (M + H)⁺ Calculated1180.1791 1194.1948 1160.2128 (m/z) (M + Na)⁺ (M + Na)⁺ (M + H)⁺ UVλ_(max) ^(MeOH) nm (ε) 213 (79833), 215 (100963), 207 (207000), 290(sh), 344 (14463) 290 (sh), 346 (29349) 347 (12000) IR ν_(max)^(KBr)cm⁻¹ 3385 (OH), 3399 (OH), 1662 (CO), 1661(CO), 1534, 1481, 7541533, 1481, 753

TABLE 2 ¹H-NMR and ¹³C-NMR Chemical Shift of QN3323-A (δ ppm)¹⁾ Position¹³C ¹H (J/Hz)  1 160.4 s —  2 148.7 s —  3 125.5 d 8.37 s  4 167.8 s — 5  57.2 d 5.46 m  5-NH — 8.32 m  6 159.7 s —  7 148.3 s —  8 124.6 d8.25 s  9 166.7 s — 10 129.3 s — 10-NH — 9.71 s 11 168.5 s — 12  56.7 d4.71 m 12-NH — 7.60 s 13 159.7 s — 14 149.8 s — 15 125.7 d 8.46 s 16164.2 s — 17 128.3 s — 18 149.6 or — 151.0 s 19 152.9 s — 20 120.6 d7.98 s 21 170.4 s — 22  56.6 d 5.05 m 22-NH 8.37 m 23 140.8 d 8.42 d(12.0) 24 118.5 d 8.33 d (6.0) 25 149.6 or — 151.0 s 26 168.2 s — 27121.6 d 8.59 s 28 149.3 s — 29 161.3 s — 30 125.4 d 8.47 s 31 150.2 s —32 159.1 s — 32-NH — 9.60 s 33 130.1 s — 34 164.4 s — 34-NH — 8.30 s 35 49.5 t 3.89 m 36 205.2 s — 37  26.8 q 2.09 s 38  66.9 d 3.97 m 39  20.9q 1.02 d (6.0) 40  71.3 s — 41  25.7 q 1.25 s 42  27.4 q 1.23 s 43 128.7d 6.50 m 44  13.6 q 1.74 d (6.0) 45  68.2 d 4.51 m 46  20.1 q 1.38 d(6.0) 47 129.0 d 6.59 m 48  13.4 q 1.73 d (6.5) 1) Measured in DMSO-d₆.¹H-NMR, 500 MHz, and ¹³C-NMR, 125 MHz.

TABLE 3 ¹H-NMR and ¹³C-NMR Chemical Shift of QN3323-B (δ ppm)¹⁾ Position¹³C ¹H (J/Hz)  1 160.4 s —  2 148.5 s —  3 126.0 d 8.41 s  4 168.2 s — 5  57.2 d 5.52 m  5-NH — 8.22 m  6 159.5 s —  7 148.1 s —  8 125.0 d8.28 s  9 166.9 s — 10 129.1 s — 10-NH — 9.53 s 11 167.6 s — 12  56.6 d4.73 m 12-NH — 7.57 s 13 159.4 s — 14 150.0 s — 15 125.4 d 8.46 s 16164.2 s — 17 128.6 s — 18 151.5 s — 19 153.8 s — 20 120.0 d 7.97 s 21170.3 s — 22  55.2 d 5.28 m 22-NH — 8.11 m 23 140.5 d 8.46 d (6.0) 24118.9 d 8.38 d (11.5) 25 149.5 s — 26 168.2 s — 27 121.6 d 8.58 s 28149.4 s — 29 161.3 s — 30 125.4 d 8.47 s 31 150.3 s — 32 159.1 s — 32-NH— 9.58 s 33 130.1 s — 34 164.4 s — 34-NH — 8.30 s 35  49.5 t 3.89 m 36205.2 s — 37  26.8 q 2.08 s 38  75.6 d 3.89 m 38-OCH₃  55.6 q 2.90 s 39 15.5 q 1.04 d (6.0) 40  71.1 s — 41  25.6 q 1.28 s 42  27.4 q 1.24 s 43128.6 d 6.49 m 44  13.9 q 1.72 d (6.0) 45  67.1 d 4.46 m 46  19.0 q 1.44d (6.5) 47 129.0 d 6.58 m 48  13.4 q 1.73 d (6.5) ¹⁾Measured in DMSO-d₆.¹H-NMR, 500 MHz, and ¹³C-NMR, 125 MHz.

From the physicochemical properties listed in Tables 1 to 3 above andFIGS. 1 and 2 below, the structures of the substances have beendetermined to have the following formulae.

QN3323-A and QN3323-B differ from thiocillin I and thiocillin II in thatthe C(36)-position in the former is carbonyl. QN3323-Y1 differs fromthiocillin I in that the double bond between C(10) and C(43) in theformer is of E-type.

EXAMPLE 3

Antibacterial Activity:

The minimal inhibitory concentration (MIC) of the compounds of theinvention obtained in Example 1, QN3323A, QN3323-B, QN3323-Y1,thiocillin I, thiocillin II and micrococcin P2 against Staphylococcusaureus FDA209P (S. aureus), Staphylococcus epidermidis IID866 (S.epidezmidis), Enterococcus faecalis IID682 (E. Faecalis) andEnterococcus faecium CAY09_(—)1 (E. faecium), as well as againstdrug-resistant bacteria, Staphylococcus aureus CAY32001 (MRSA) (S.aureus (MRSA)) and Enterococcus faecium CAY09_(—)2 (VRE) (E. faecium(VRE)) was measured according to the standard method of the ChemotherapySociety of Japan (CHEMOTHERAPY, 1981, 29(1):76, revised minimalinhibitory concentration (MIC) determination method). The cells of S.aureus (MRSA) were incubated at 32° C. The data obtained are shown inTable 4.

TABLE 4 MIC (μg/ml) QN3323-A QN3323-B QN3323-Y1 Thiocillin I ThiocillinII Micrococcin P2 S. aureus 0.05 0.20 0.25 S. epidermidis 0.39 0.20 0.25E. faecalis 1.56 0.78 0.5 E. faecium 1.56 0.78 0.5 S. aureus (MRSA) 1.561.56 2 0.78 0.10 0.78 E. faecium (VRE) 1.56 0.20 0.25 0.78 0.05 0.2

EXAMPLE 4

Remedial Effect for Infected Mouse Model

The compounds of the invention, QN3323-A, QN3323-B, thiocillin I andthiocillin II were tested using an infected mouse model to determine theeffectivness thereof. Concretely, cells of Staphylococcus aureus Smithcultivated for 16 hours at 37° C. were suspended in 3% butamutin (byTokyo Kasei), and the resulting cell suspension was intraabdominallyinfected into 4-week old male ICR(CD-1) mice (from Charles River ofJapan, SPF) (about 3×10⁶ CFU/mouse). Two hours after the infection, thecompound of the invention dissolved in a solvent (10% DMSO, 10% HCO-60(by Nikko Chemicals), 80% physiological saline), was subcutaneouslyadministered to each mouse. On 4 to 7 days after the treatment, the micewere observed, and their ED50 value was calculated according to probitmethod. The data are given in Table 5.

TABLE 5 ED5O (mg/kg) QN3323-A 7.6 QN3323-B 13.2 Thiocillin I 1.4Thiocillin II 2.2

From the above, it is understood that the novel thiopeptide compounds(I′) of the invention have good antibacterial activity especially evenagainst multidrug-resistant bacteria such as MRSA and VRE, and thereforethe compounds are useful for medicines, especially for antibacterialagents, more particularly for preventives and remedies for infectionswith multidrug-resistant bacteria. In addition, known compounds,thiocillin I, thiocillin II and micrococcin P2 have good antibacterialactivity against MRSA and VRE, and are useful for preventives andremedies for infections with multidrug-resistant bacteria, especiallyfor MRSA or VRE infections.

1. Thiopeptide compounds of the following formula or theirpharmaceutically acceptable salts:

wherein A represents CH—OH or C═O; R represents H or CH₃; R^(a) andR^(b) each represent H or CH₃; provided that when R^(a) is H, then R^(b)is CH₃; when R^(a) is CH₃, then R^(b) is H; when A is CH—OH, then R^(a)is H and R^(b) is CH₃.
 2. Compounds or their pharmaceutically acceptablesalts as claimed in claim 1, wherein A is C═O, R is H, R^(a) is CH₃, andR^(b) is H.
 3. Compounds or their pharmaceutically acceptable salts asclaimed in claim 1, wherein A is C═O, R is CH₃, R^(a) is CH₃, and R^(b)is H.
 4. Compounds or their pharmaceutically acceptable salts as claimedin claim 1, wherein A is CR—OH, R is H, R^(a) is H, and R^(b) is CH₃. 5.A pharmaceutical composition that comprises, as the active ingredientthereof, a compound of claim 1 or its pharmaceutically acceptable saltand a pharmaceutically acceptable carrier.
 6. The pharmaceuticalcomposition as claimed in claim 5, which is an antibacterial agent.
 7. Amethod for producing thiopeptide compounds of formula (I′), whichcomprises cultivating cells of Bacillus sp. QN03323 (FERM BP-7864)followed by collecting the thiopeptide compounds of claim 1 from theresulting culture.
 8. A method for treating infections caused bymultidrug-resistant bacteria, which comprises administering to a patientin need thereof an effective amount of a thiopeptide compound of thefollowing formula I′ or a pharmaceutically acceptable salt thereof:

wherein A represents CH—OH or C═O; R represents H or CH₃; R^(a) andR^(b) each represent H or CH₃; provided that when R^(a) is H, then R^(b)is CH₃, when R^(a) is CH₃, then R^(b) is H; when A is CH—OH, then R^(a)is H and R^(b) is CH₃.
 9. The method as claimed in claim 8, which is fortreating infections caused by methicillin-resistant Staphylococcusaureus or vancomycin-resistant Enterococcus.